Apparatus for preparing encapsulated bodies

ABSTRACT

An apparatus for the production of encapsulated bodies employing a dropping tank for a fluid to form the cores of the encapsulated bodies and a reaction tank containing a solution capable of forming gel skins upon contact with the core material in communication therewith. Double-walled cylindrical nozzles are arranged in a lower part of the drop tank, the inner and outer walls of the double-walled cylindrical nozzles being coaxial. A pressurized air feed pipe is in communication with an upper part of the dropping tank. The peripheral walls of the reaction tank are in the form of a double-walled cylinder whose upper peripheral portion of its inner wall contains a screen. A liquid feed pipe and a capsule discharge port are provided in communication with the interior of the reaction tank through lower parts of the double-walled cylinder. A liquid discharge port also is provided in communication with an annular spacing of the double-walled cylinder through its outer wall. 
     The apparatus provides for obtaining a dispersion of encapsulated bodies without their mechanical separation from the liquid in which they are prepared.

This is a division of application Ser. No. 07/009,926, filed Feb. 2,1987, now U.S. Pat. No. 4,814,274.

BACKGROUND OF THE INVENTION

1) Field of the Invention

This invention relates to a process for producing encapsulated bodies byinducing gelation through a liquid-liquid reaction, in which theresulting encapsulated bodies are recovered in a form dispersed in adesired liquid without mechanical separation of the resultingencapsulated bodies from the reaction mixture, and to an apparatustherefor.

2) Description of the Prior Art

In recent years, encapsulation of various liquid materials is practicedin many fields in order to immobilize or otherwise to fix the liquidmaterials.

For example, there are jelly foods, artificial roe, encapsulateddressing and encapsulated fruit juice in the field of foods.

It is also interested recently in immobilizing microorganisms andenzymes by using capsules or gel particles or granules in the field ofso-called biotechnology, especially, in the microencapsulation techniqueas a method for culturing animal cells.

Incidentally, the culture making use of the aforementionedmicroencapsulation technique permits protection of cells from mechanicalshear forces during the culture. It is also feasible to allow aphysiologically active substance, which is to be produced by the cells,to accumulate to a high concentration within the capsules by controllingthe permeability of the capsule walls, resulting in the advantages thatnot only the isolation and recovery of the physiologically activesubstance but also the separation of the cells and the culture mediumare facilitated.

The above-described encapsulation technique is however accompanied by aproblem upon its utilization, because damages to the capsules or gelparticles formed by the gelation, such as their rupture, are unavoidabledue to an application of mechanical separation such as filtration orcentrifugation for the separation and recovery of the capsules or gelparticles from the reaction mixture. In the case of encapsulation ofanimal cells or the like, the resultant capsules must be transferred ina sterile state promptly to a culture medium and then to disperse samethere. The above-mentioned mechanical separation hence involves theproblem that the sterilization procedure becomes very complex.

SUMMARY OF THE INVENTION

This invention has been completed in view of the above-describedproblems in the production of encapsulated bodies. The present inventorshave succeeded in recovering capsules (gel particles), which have beenformed through gelation by a liquid-liquid reaction, in a form dispersedin a liquid desired for the utilization of the capsules without damagingthe capsules by effecting the separation and recovery of the capsulesfrom the reaction mixture in accordance with a method, in which theliquid portion of the reaction mixture is substituted by the desiredliquid, instead of using mechanical separation means.

An object of this invention is therefore to provide a process forrecovering, without using mechanical means, encapsulated bodies (e.g.,capsules or gel particles), which have been formed by gelation through aliquid-liquid reaction, in a form dispersed in a desired liquid from thereaction mixture in which the encapsulated bodies are dispersed.

In one aspect of this invention, there is thus provided a process forthe production of encapsulated bodies by bringing a fluid, which is toform the cores of the encapsulated bodies, and a solution, which hasability to form gel skins upon contact with the fluid, into contact andinducing a gelling reaction therebetween, which comprises introducing adesired liquid, which is suitable for the utilization of theencapsulated bodies, successively into the reaction mixture in which theencapsulated bodies formed by the gelling reaction are dispersed,whereby the liquid portion of the reaction mixture is substituted withthe desired liquid and the resultant encapsulated bodies are hencerecovered in a form dispersed in the desired liquid.

In another aspect of this invention, there is also provided an apparatusfor the production of encapsulated bodies, including a dropping tank fora fluid to be employed to form the cores of the encapsulated bodies anda reaction tank for containing a solution capable of forming gel skinsupon contact with the fluid, characterized in that double-walledcylindrical nozzles are arranged in a lower part of the dropping tank,the inner and outer walls of each of the double-walled cylindricalnozzles are coaxial, a pressurized air feed pipe is provided incommunication with an upper part of the dropping tank, the peripheralwall of said reaction tank is constructed in the form of a double-walledcylinder, an upper peripheral portion of the inner wall of thedouble-walled cylinder is formed in the shape of a screen, a liquid feedpipe and a capsule discharge port are provided in communication with theinterior of the reaction tank through lower parts of the double-walledcylinder, and a liquid discharge port is formed in communication withthe annular spacing, which is defined by the inner and outer walls ofthe double-walled cylinder, through the outer wall of the double-walledcylinder.

The term "encapsulated bodies" as used herein means not only capsulesbut also gel particles. The term "desired liquid" will hereinafter bereferred to as "dispersing medium" for the sake of simplicity.

The present invention has brought about numerous advantages. Accordingto the present invention, the encapsulated bodies formed by the gellingreaction can be recovered in a form dispersed in a dispersing mediumsuitable for the utilization of the thus-encapsulated bodies withoutusing mechanical separation means such as filtration or centrifugation.It is therefore possible to obtain the encapsulated bodies in a formconforming with their application purpose without damaging them. Inaddition, the use of the apparatus of this invention allows to conductthe above-mentioned recovery of the encapsulated bodies and at the sametime, to control the size of the encapsulated bodies as desired.Further, the encapsulated culture of animal cells or the like permitstheir handling in a sterile state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side cross-sectional view showing the outline of aproduction apparatus according to the present invention; and

FIG. 2 is a transverse cross-sectional view taken along line A--A inFIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

As reactant solutions useful for gelation in the production ofencapsulated bodies in accordance with this invention, the followingcombinations may be mentioned by way of example: Ca⁺⁺ containingsolution (core liquid)--a solution of sodium alginate or alowly-methoxylated pectin; and an aqueous solution (core liquid) of apolyanionic polysaccharide such as carboxymethylcellulose or its saltand a solution of chitosan. The latter combination is particularlysuitable for the culture of animal cells by the microencapsulationtechnique, because the gelation can be carried out under mildconditions.

The contact of the reactant solutions in each of the above-mentionedcombinations can be effected usually by dropping one of the reactantsolutions (for example, a Ca⁺⁺ containing solution,carboxymethylcellulose solution) as a core liquid, into the otherreactant solution.

In the present invention, a gelling reaction is induced between bothreactant solutions as a result of the dropwise contact of one of thereactant solutions with the other reactant solution so that capsules (orgel particles) are formed and dispersed in the reaction mixture. Bysubstituting the liquid portion of the reaction mixture with adispersing medium suitable for the utilization of the resultantcapsules, the capsules are recovered in a form dispersed in thedispersing medium.

A method, which is useful for the recovery of the resulting capsules ina form dispersed in the dispersing medium in accordance with thisinvention, will next be described on the basis of an apparatus usable inthe practice of the above method.

Reference is first made to FIG. 1. Numeral 1 indicates a dropping tankfor a core liquid, whereas numeral 2 designates a reaction tank forstoring a liquid which undergoes a gelling reaction with the core liquidupon their mutual contact. The dropping tank 1 has double-walledcylindrical nozzles 3 in a lower part thereof and a pressurized gas feedline 4 in an upper part thereof. The inner and outer walls of each ofthe nozzles 3 are coaxial.

There are also shown a core solution inlet 5, an air filter 6 forremoving bacteria from a pressurized gas to be introduced, a vent pipe 7for the pressurized gas, and a valve 8 for the vent pipe. Designated atnumeral 9 is a feed pipe for another pressurized gas to be introducedinto annular spacings between the inner and outer walls of each of thedouble-walled cylindrical nozzles 3 in order to control the size ofdroplets of the core solution to be dropped through the nozzles 3. Thefeed pipe 9 is therefore in communication with the annular spacings. Thethus-introduced gas flows out through the annular spacings and thenalong the imaginary extensions of the inner walls of the nozzles 3,thereby controlling the size of the droplets of the core solution.Numeral 10 indicates an air filter for making the pressurized gas freeof bacteria.

On the other hand, the peripheral wall of the reaction tank 2 is formedin the shape of double cylinders. An upper part of the inner peripheralwall of the reaction 2 is formed as a screen-like portion 11. Adispersing medium feed pipe 12 and capsule discharge pipe 13 areprovided in lower parts of the reaction tank 2. The dispersing mediumfeed pipe 12 and capsule discharge pipe 13 are in communication with theinterior of the reaction tank 2 through both inner and outer walls ofthe double-walled cylindrical peripheral wall of the reaction tank 2.Through the outer wall of the peripheral wall, a liquid outlet 14 isalso provided in communication with the annular spacing between theinner and outer walls of the peripheral wall.

In the same drawing, numerals 15, 16 indicate valves, numeral 17 amagnetic stirrer provided in the bottom portion of the reaction tank 2,and numeral 18 a rotating electromagnet.

In the present invention, a core solution which is to form the cores ofencapsulated bodies is prepared first of all. The core solution ischarged through the charging opening 5 into the dropping tank 1. On theother hand, the reaction tank 2 is filled with a solution whichundergoes a gelling reaction with the core liquid. When animal cells orthe like are encapsulated and cultured, it is necessary to maintain theinterior of the apparatus under sterile conditions. Since the droppingtank and reaction tank are provided in continuation in the apparatus,the solution for conducting a gelling reaction is introduced through adispersing medium feed pipe 12 provided at a lower part of the reactiontank 2.

Pressurized air or the like is then introduced through the pressurizedgas feed pipe 4 provided in the upper part of the dropping tank 1 so asto pressurize the core solution, whereby the core solution is droppedinto the reactant solution in the reaction tank 2 through the nozzles 3.Another pressurized air or the like is fed through the pressurized gasfeed pipe 9 provided through the outer peripheral wall of the reactiontank 2. This pressurized air or the like is then allowed to flow alongthe imaginary extensions of the inner walls of the nozzles 3, therebycontrolling the size of droplets of the core solution to achieve adesired capsule size.

The thus-dropped core solution is thus brought into contact with thereactant solution in the reaction tank 2 to undergo a gelling reactionimmediately, so that the droplets of the core solution are convertedinto capsules or gel particles. Upon conducting the gelling reaction, itis possible to choose stirring conditions, which are most suitable foreach one of various encapsulation methods, by using the magnetic stirrer17 placed in the bottom part of the reaction tank 2 and then to stir themixture in the reaction tank 2.

Upon completion of the above-described encapsulation, the valve 15 ofthe dispersing medium feed pipe 12 is opened so as to introduce adispersing medium, which is suitable for the utilization of the capsules(for example, diluting water, fruit juice, syrup, liquid culturemedium), under pressure through the feed pipe 12. Thus, the dispersingmedium is introduced successively into the reaction tank 2. Owing tothis introduction of the dispersing medium, the liquid level rises inthe reaction tank 2. Since the upper part of the inner peripheral wallof the reaction tank 2 is formed as the screen-like portion 11, anyextra portion of the liquid is caused to overflow to maintain thepredetermined liquid level in the reaction tank 2. The extra portion ofthe liquid is then allowed to flow down through the spacing between theinner and outer walls of the double-walled cylinder which constitutesthe reaction tank 2, whereby the extra portion of the liquid issuccessively discharged out of the reaction tank 2 through the liquidoutlet 14 formed through the outer wall. Here, the capsules dispersed inthe reaction mixture are not overflowed owing to the provision of thescreen-like portion. Accordingly, the concentration of the reactionmixture in the reaction tank 2 drops exponentially at a ratecommensurate with the amount of the dispersing medium introduced underpressure through the feed pipe 12. Upon a lapse of a certain period oftime, the liquid portion of the reaction mixture has been substitutedwith the above-introduced dispersing medium.

In passing, the above-mentioned exponential relation between theconcentration of the reaction mixture in the reaction tank and time canbe expressed by the following equation. ##EQU1## where C_(t) . . .Concentration of the reaction mixture after t hours

C_(o) . . . Initial concentration of the reaction mixture

Q . . . Flow rate of the introduced dispersing medium

V . . . Volume of the reaction tank

t . . . Introduction time of the dispersing medium

At a stage where the reaction mixture in the reaction tank 2 has beensubstituted practically with the introduced dispersing medium in themanner mentioned above (at this stage, the resultant capsules are eitherdispersed or suspended in the thus-introduced dispersing medium), thevalve 15 of the feed pipe 12 for the dispersing medium is closed and thevalve 16 of the capsule discharge pipe 13 is opened so as to dischargeand recover the capsules together with the dispersing medium from thereaction tank 2.

When conducting encapsulated culture of animal cells or the like in thepresent invention, a physiological saline or phosphate-buffered saline(PBS) is introduced in the reaction tank so as to substitute for theliquid portion of the reaction mixture in the reaction tank. Thecapsules, which contain the animal cells as a core substance, are thenrecovered in a form dispersed in PBS or the like. The capsule dispersionis thereafter mixed with an equal amount of a culture medium which hasbeen prepared in advance with a concentration twice its usualconcentration, followed by culture of the animal cells.

Encapsulated bodies dispersed in a syrup or fruit juice may be obtainedby introducing water into a reaction mixture as a dispersing medium atthe time of the encapsulation reaction being completed in order todilute the mixture and then introducing the syrup or fruit juice as themedium thereby recovering capsulated bodies dispersed in the syrup orfruit juice.

The present invention will hereinafter be described more specifically bythe following Examples.

EXAMPLE 1

This example illustrates encapsulation of animal cells. A liquid culturemedium containing 0.5% of carboxymethylcellulose (CMC), in which a IgGantibody producing hybridoma had been dispersed at a concentration of2×10⁵ cells/ml, was employed as a core solution. The core solution wascharged into the dropping tank 1 of the production apparatus, which isdepicted by way of example in FIG. 1, through the core solution inlet 5.The core solution was then dropped under pressure through the nozzles 3,which were provided in the lower part of the dropping tank, formed asdouble-walled cylinders with the inner and outer walls thereof beingcoaxial and had an inner diameter of 0.5 mm, into a 0.7% chitosansolution received in the reaction tank 2. The pressurization of the coresolution was effected by introducing air under pressure from thepressurized gas feed pipe 4, which was provided in the upper part of thedropping tank, through the air filter into the dropping tank. At thesame time, air was also fed from the pressurized gas feed pipe 9, whichwas connected to the outer walls of the double-walled cylindricalnozzles 3, through the air filter 10 into the annular spacings betweenthe outer and inner walls of the nozzles and was then caused to flow outalong the imaginary extensions of the inner walls of the respectivenozzles, so that the diameter of the resulting droplets of the coresolution was controlled at 2 mm.

The above core solution dropped in the reaction tank 2 was brought intocontact with the chitosan solution to induce a gelling reaction. Two tothree minutes later, capsules were formed and dispersed in the reactionmixture.

The valve 15 was thereafter opened to successively introduce PBS as adiluent through the dispersing medium feed pipe 12 provided in the lowerpart of the reaction tank 2, thereby substituting the chitosan solutionwith PBS.

After the above substitution, the capsules dispersed in PBS wererecovered through the capsule discharge pipe provided in the lower partof the reaction tank and then fed to a culture tank, in which the PBSdispersion of the capsules was mixed with an equal amount of a culturemedium ["DMEM" (Dulbecco's Modified Eagle's Medium; trade name) and"Ham's F12" (trade name) were used] which had been prepared in advancewith a concentration twice its usual concentration, followed by cultureof the encapsulated cells.

EXAMPLE 2

This example illustrates encapsulation of a fruit juice.

A core solution was prepared by adding 4 parts by weight offivefold-concentrated strawberry juice to 100 parts by weight of asorbitol solution which contained 3% of calcium lactate. Following theprocedure of Example 1, the core solution was added dropwise with adroplet size of 6 mm into a 2.5% solution of lowly-methoxylated pectinso that the core solution was encapsulated.

Three minutes after the encapsulating reaction, a sorbitol solution of"Brix45" was introduced successively into the reaction tank in the samemanner as in Example 1. The reaction mixture in the reaction tank wassubstituted with the sorbitol solution and the encapsulated fruit juicewas recovered in a form dispersed in the sorbitol solution.

EXAMPLE 3

As a core solution, there was used a yeast dispersion which had beenobtained by dispersing yeast at a concentration of 20% (w/v) in aphysiological saline containing 4% of κ-carrageenan. Following theprocedure of Example 1, the core solution was dropped with a particlesize of 4 mm in a 400 mM aqueous solution of potassium chloride, therebysubjecting the droplets of the core solution to a gelling reaction.

Upon a lapse of three minutes from the completion of the gellingreaction, a physiological saline was introduced successively into thereaction tank so as to substitute, with the physiological saline, theliquid portion of the reaction mixture in the reaction tank. Theresultant encapsulated yeast was then recovered in a form dispersed inthe physiological saline.

What is claimed is:
 1. An apparatus for the production of encapsulatedbodies, comprising a dropping tank for a fluid to be employed to formthe cores of the encapsulated bodies and a reaction tank having aperipheral inner and outer wall for containing a solution forming gelskins upon contact with said fluid in communication therewith,double-walled cylindrical nozzles arranged in a lower part of thedropping tank, the inner and outer walls of each of said double-walledcylindrical nozzles being coaxial, a pressurized air feed pipe incommunication with an upper part of said dropping tank, the peripheralwalls of said reaction tank comprising a double-walled cylinder whoseupper peripheral portion of the inner wall of said double-walledcylinder comprises a screen, a liquid feed pipe and a capsule dischargeport in communication with the interior walls of said reaction tankthrough lower parts of said double-walled cylinder, and a liquiddischarge port in communication with an annular spacing defined by theinner and outer walls of said double-walled cylinder through the outerwall of said double-walled cylinder, the interior of said reaction tankbeing in fluid communication with said annular space through saidscreen.
 2. The apparatus as claimed in claim 1, further comprising astirring means in a bottom part of the reaction tank for stirring theencapsulated bodies.
 3. The apparatus as claimed in claim 1, furthercomprising a pressurized gas feed pipe in communication with annularspacings of the double-walled cylindrical nozzles, said annular spacingsbeing defined by the inner and outer walls of the respectivedouble-walled cylindrical nozzles.